笋用林3种竹笋夜蛾多重PCR鉴定技术的建立与应用  

Development and Application of Multiplex PCR Assay for Identification of Three Noctuid Species in Shoot-used Bamboo Forests

作　　者：耿显胜[1] 赵誉霞 贾小琦 彭嫔嫔 张威[1] 舒金平[1] Geng Xiansheng;Zhao Yuxia;Jia Xiaoqi;Peng Pinpin;Zhang Wei;Shu Jinping(Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Hangzhou 311400;Zhejiang Linxin Forestry Technology Service Co.,Ltd,Hangzhou 311400)

机构地区：[1]中国林业科学研究院亚热带林业研究所,杭州311400 [2]浙江林信林业技术服务有限公司,杭州311400

出　　处：《林业科学》2024年第10期86-93,共8页Scientia Silvae Sinicae

基　　金：浙江省科技计划项目公益技术应用研究类(LGN21C160011)。

摘　　要：【目的】建立鉴定竹笋夜蛾物种的多重PCR技术,用于浙江省笋用竹的3种竹笋夜蛾幼虫的物种鉴定。【方法】针对COI基因的变异区设计物种特异性多重PCR引物;优化影响多重PCR反应的参数,建立鉴定竹笋夜蛾的多重PCR技术;采用标准DNA模板评价多重PCR技术的敏感性和特异性;利用多重PCR技术对林间采集的竹笋夜蛾幼虫进行物种鉴定。【结果】针对3种竹笋夜蛾COI基因的变异区设计了3条物种特异性多重PCR引物,与通用引物LCO1490配合使用,扩增竹笋基夜蛾、竹笋禾夜蛾和笋秀夜蛾COI基因片段,大小分别为290、390和590 bp。优化后的多重PCR鉴定技术的反应体系为:2×HotStart Taq PCR预混试剂10μL,10μmol·L^(-1)的引物LCO1490、JYE290、HYE390和QTYE590各0.5μL,DNA模板1μL,加ddH2O补足20μL。优化后的多重PCR鉴定技术的反应条件为:94℃预变性5 min;94℃变性30 s,59℃退火30 s,72℃延伸30 s;72℃下延伸10 min,循环数为35。多重PCR鉴定技术对笋秀夜蛾的最低检出限为0.01 ng·μL^(-1),对竹笋禾夜蛾和竹笋基夜蛾的最低检出限低于0.001 ng·μL^(-1)。林间样品鉴定结果表明,所有38份DNA都能扩增出明显的特异性条带,鉴定成功率100%;经测序验证,本研究的技术鉴定的物种与经COI基因序列鉴定的物种一致。【结论】研究建立了浙江省笋用竹的夜蛾的多重PCR鉴定技术,能够快速高效鉴定浙江省笋用林内竹笋夜蛾幼虫的物种,该技术具有鉴定周期短、灵敏度高、特异性强、准确度高等优点。【Objective】This study aims to develop a multiplex PCR assay for identification of larvae of three noctuid moth species feeding on bamboo shoots in Zhejiang Province.【Method】Species-specific multiplex PCR primers were designed for COI gene variation regions.The parameters influencing the multiplex PCR reaction were optimized,and the multiplex PCR assay was developed for identification of noctuids.The sensitivity and specificity of the proposed multiplex PCR assay were determined using standard templates.Species identification of larvae of noctuids collected from shoot-used bamboo forests was performed by the proposed multiplex PCR assay.【Result】In the study,three species-specific multiplex PCR primers were designed for variation regions of COI gene in three noctuid species,and these primers were used in conjunction with the universal primer LCO1490 to amplify fragments of the COI genes of Kumasia kumaso,Oligia vulgaris,and Apamea apameoides with the sizes of 290,390,and 590 bp,respectively.The optimized multiplex PCR was performed in a 20μL reaction mixture including 10μL 2×HotStart Taq PCR premix reagent,0.5μL(10μmol·L^(-1))each of primers LCO1490,JYE290,HYE390 and QTYE590,1μL DNA template,and added ddH2O to 20μL.The reaction conditions for the optimized multiplex PCR were 94℃for 5 min followed by 35 cycles of 94℃for 30 s,59℃for 30 s,and 72℃for 30 s,followed by a final extension for 10 min at 72℃.The minimum detection limit of proposed multiplex PCR assay was 0.01 ng·μL^(-1) for A.apameoides,and less than 0.001 ng·μL^(-1) for K.kumaso and O.vulgaris.The identification results of larvae of noctuids collected from shoot-used bamboo forests showed that all 38 samples were able to amplify obvious specific bands,and the success rate of the multiplex PCR assay was 100%.Moreover,the species identified according to the technique in this study were completely consistent with the species identified by the COI gene sequence.【Conclusion】A multiplex PCR assay for identification of larvae

关 键 词：雷竹 夜蛾 引物 多重PCR DNA条形码 

分 类 号：S763.302[农业科学—森林保护学]